Oncolytic Virus Strain

ABSTRACT

The present invention relates to an oncolytic virus which is, or is derived from, a clinical isolate which has been selected by comparing the abilities of a panel of three or more clinical isolates of the same viral species to kill tumor cells of two or more tumor cell lines in vitro and selecting a clinical isolate which is capable of killing cells of two or more tumor cell lines more rapidly and/or at a lower dose in vitro than one or more of the other clinical isolates in the panel.

FIELD OF THE INVENTION

The invention relates to an oncolytic immunotherapeutic agent and to theuse of the oncolytic immunotherapeutic agent in treating cancer.

BACKGROUND TO THE INVENTION

Viruses have a unique ability to enter cells at high efficiency. Afterentry into cells, viral genes are expressed and the virus replicates.This usually results in the death of the infected cell and the releaseof the antigenic components of the cell as the cell ruptures as it dies.As a result, virus mediated cell death tends to result in an immuneresponse to these cellular components, including both those derived fromthe host cell and those encoded by or incorporated into the virusitself.

Viruses also engage with various mediators of the innate immune responseas part of the host response to the recognition of a viral infectionthrough e.g. toll-like receptors and cGAS/STING signalling resulting inthe activation of interferon responses and inflammation which are alsoimmunogenic signals to the host. These immune responses may result inthe immunogenic benefit to cancer patients such that immune responses totumor antigens provide a systemic overall benefit resulting in thetreatment of tumors which have not been infected with the virus,including micro-metastatic disease, and providing vaccination againstrelapse.

The combined direct (‘oncolytic’) effects of the virus, and immuneresponses against tumor antigens (including non-self ‘neo-antigens’,i.e. derived from the particular mutated genes in individual tumors) istermed ‘oncolytic immunotherapy’.

Viruses may also be used as delivery vehicles (‘vectors’) to expressheterologous genes inserted into the viral genome in infected cells.These properties make viruses useful for a variety of biotechnology andmedical applications. For example, viruses expressing heterologoustherapeutic genes may be used for gene therapy. In the context ofoncolytic immunotherapy, delivered genes may include those encodingspecific tumor antigens, genes intended to increase the immunogenicityof antigens released following virus replication and cell death, toincrease the general immune activation status of the tumor, or toincrease the direct oncolytic properties (i.e. cytotoxic effects) of thevirus.

It has been demonstrated that a number of viruses including herpessimplex virus (HSV) have utility in the oncolytic treatment of cancer.HSV for use in the oncolytic treatment of cancer must be disabled suchthat it is no longer pathogenic, but can still enter into and kill tumorcells. A number of disabling mutations to HSV, including disruption ofthe genes encoding ICP34.5, ICP6, and/or thymidine kinase, have beenidentified which do not prevent the virus from replicating in culture orin tumor tissue in vivo, but which prevent significant replication innormal tissue. HSVs in which only the ICP34.5 genes have been disruptedreplicate in many tumor cell types in vitro, and replicate selectivelyin tumor tissue, but not in surrounding tissue, in mouse tumor models.Clinical trials of ICP34.5 deleted, or ICP34.5 and ICP6 deleted, HSVhave also shown safety and selective replication in tumor tissue in man.

As discussed above, an oncolytic virus, including HSV, may also be usedto deliver a therapeutic gene in the treatment of cancer. An ICP34.5deleted virus of this type additionally deleted for ICP47 and encoding aheterologous gene for GM-CSF has also been tested in clinical trials,including a phase 3 trial in melanoma in which safety and efficacy inman was shown. The trial data demonstrated that tumor responses could beseen in injected tumors, and to a lesser extent in uninjected tumors.Responses tended to be highly durable (months-years), and a survivalbenefit appeared to be achieved in responding patients. Each of theseindicated engagement of the immune system in the treatment of cancer inaddition to the direct oncolytic effect. However, this and other datawith oncolytic viruses generally showed that not all tumors respond totreatment and not all patients achieve a survival advantage. Thus,improvements to the art of oncolytic therapy and oncolytic immunotherapyare clearly needed. These may serve to increase the direct oncolyticeffects of therapy, the anti-tumor immune stimulating effects of thetherapy, or both of these effects together.

Recently it has been shown that oncolytic immunotherapy can result inadditive or synergistic therapeutic effects in conjunction with immunecheckpoint blockade (i.e. inhibition or ‘antagonism’ of immunecheckpoint pathways), also referred to as immune co-inhibitory pathwayblockade. Checkpoint (immune co-inhibitory pathway) blockade is intendedto block host immune inhibitory mechanisms which usually serve toprevent the occurrence of auto-immunity. However, in cancer patientsthese mechanisms can also serve to inhibit or block the potentiallybeneficial effects of any immune responses induced to tumors.Alternatively, immune responses may not be fully potentiated due to alack of activation or lack of full activation of immune potentiatingpathways. Therefore, drugs which alleviate these blocks or stimulateimmune potentiating pathways (i.e. which activate, or are ‘agonists’ ofthese immune potentiating pathways) are attractive for testing anddeveloping cancer treatments. Targets for such approved or experimentaldrugs include CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, VISTA, CSF1R, IDO,CEACAM1, GITR, 4-1-RB, KIR, SLAMF7, OX40, CD40, ICOS or CD47.

For these approaches to be successful, pre-existing immune responses totumors are needed, i.e. so that a pre-existing immune response can bepotentiated or a block to an anti-tumor immune response can be relieved.The presence of an inflamed tumor micro-environment, which is indicativeof such an ongoing response, is also needed. Pre-existing immuneresponses to tumor neo-antigens appear to be particularly important forthe activity of immune co-inhibitory pathway blockade and related drugs.Only some patients may have an ongoing immune response to tumor antigensincluding neoantigens and/or an inflamed tumor microenvironment, both ofwhich are required for the activity of these drugs. Therefore, oncolyticagents which can induce immune responses to tumor antigens, includingneoantigens, and/or which can induce an inflamed tumor microenvironmentare attractive for use in combination with immune co-inhibitory pathwayblockade and immune potentiating drugs. This likely also explains thepromising combined anti-tumor effects of oncolytic agents and immuneco-inhibitory pathway blockade in mice and humans that have so far beenobserved.

The indoleamine 2,3-dioxygenase (IDO) pathway contributes totumor-induced tolerance by creating a tolerogenic environment in thetumor and the tumor-draining lymph nodes, both by direct suppression ofT cells and enhancement of local regulatory T cell (Treg)-mediatedimmunosuppression. IDO catalyses the rate-limiting step of tryptophandegradation along the kynurenine pathway, and both the reduction inlocal tryptophan concentration and the production of immunomodulatorytryptophan metabolites contribute to the immunosuppressive effects ofIDO. IDO is chronically activated in many cancer patients with IDOactivation correlating with more extensive disease. It can also functionas an antagonist to other activators of antitumor immunity. Therefore,inhibitors of the IDO pathway are being developed as anticancer agents,particularly in combination with checkpoint blockade agents such asthose which target CTLA-4, PD-1 or PDL-1. IDO inhibitors may also besynergistic with oncolytic immunotherapy, including together with drugstargeting other immune checkpoint or immune co-stimulatory pathways.

SUMMARY OF THE INVENTION

The invention provides improved oncolytic viruses. The improvedoncolytic viruses have improved direct oncolytic effects. The improveddirect oncolytic effects provided by the viruses of the invention willalso lead to improved systemic anti-tumor immune effects. The improveddirect oncolytic effects provided by the viruses of the invention willalso lead to improved therapeutic effects in patients. Enhancedreplication in and killing of tumor cells will result in enhanced tumorantigen release and enhanced systemic immune responses to the releasedantigens. The expression levels of any genes inserted to augment thedirect oncolytic effects and/or immune stimulation will also beincreased.

Virus species naturally exist in a range of variants (strains) withinthe natural population which may differ by a small or larger number ofnucleotides while still retaining the antigenic characteristics andsufficient sequence identity to still be recognized as the same virusspecies. These strains, due to their differing sequences, may exhibit arange of differing properties, including properties which have beenselected for by natural selection in their natural host or hosts (forexample the ability to infect or replicate in the target cell types ofthe virus in question, spread between these cells, or to evade the hostinnate or adaptive immune system, or to spread between infectedindividuals of the host species) and properties which have not beenspecifically selected for (e.g. the ability to replicate in and kill orspread between cell types which are not the natural targets of the virusin question, including tumor or other non-target cell types or tissues).The inventors have recognised that sampling a range of viral strains ofa particular viral species which are present in the natural hostpopulation (in the case of viruses infecting humans, here termed‘clinical isolates’) and comparing these to each other to select for thestrain with the best properties for the intended purpose for which it isto be used (e.g. infection and killing of tumor cells) can be used toidentify a virus (i.e. a virus strain) with optimal properties for thatpurpose. The optimal properties may be properties that offer the beststarting point for development to produce a virus that can be used as atherapeutic. A virus identified by this approach is likely to have moreoptimal properties for the intended purpose than a ‘prototype’ or‘laboratory’ virus strain or a clinical strain which has not beenselected for the required property or properties from a broad group ofviral strains. This is because the full biological complexity in thenatural population, particularly with respect to the particulardesirable property or properties, is unlikely to have been sampledthrough taking a narrow approach to screening for the desired propertyor properties, bearing in mind the degree of sequence variation presentin natural virus populations. In particular, these may vary in sequencewithin an infected host (as is often the case with RNA or retroviralpopulations where so-called quasi-species are often present), betweenindividual infected hosts, or between different geographically separatedviral populations.

Viruses of the invention have therefore been selected by sampling arange of viral strains present in the natural population of a particularviral species and testing these against each other for the desiredproperty or properties (e.g. the ability to infect and kill tumorcells). The virus strain or strains with the best properties for theintended purpose are used for further development.

Where the intended use is oncolytic viral therapy, taking such anapproach provides an improved starting point for development of anoncolytic agent, which may require further manipulation of theadvantageous virus strains. Such manipulation includes the deletion ofviral genes to provide, for example, tumor selectivity, and/or theinsertion of exogenous genes to improve oncolytic or immune potentiatingproperties further.

The viruses of the invention therefore include novel clinical isolatesof a viral species that have better anti-tumor effects than the otherclinical isolates to which they were compared and through whichcomparison they were identified. In particular, the clinical isolates ofthe invention kill tumor cell lines in vitro more quickly and/or at alower dose than these reference clinical isolates of the same virustype. Typically, a clinical isolate of the invention will have beenidentified through comparison of >5 clinical isolates of a viral speciesfor the required property or properties, preferably through comparisonof >10 clinical isolates of the viral species, and more preferablythrough comparison of >20 clinical isolates of the viral species. Aclinical isolate of the invention typically shows better tumor cellkilling activity than ⅗, 6/10 or 11/20ths, preferably better than ⅘,8/10 or 17/20ths, more preferably better than 9/10 or 19/20ths of theviral strains tested.

Typically, a clinical isolate of the invention can kill two or moretumor cell lines in vitro within 24 to 48 hours after infection at amultiplicity of infection (MOI) of 0.01 to 0.001 or less.

The clinical isolates of the invention may be modified to furtherenhance their anti-tumor effects. The genome of a clinical isolate ofthe invention may be modified to delete or alter expression of one ormore viral genes, and/or the genome of the clinical isolate may bemodified to express one or more heterologous genes, such as genesencoding a fusogenic protein and/or an immune stimulatory molecule ormolecules.

Oncolytic viruses of the invention provide improved treatment of cancerthrough improved direct oncolytic effects, viral replication and spreadthrough tumors, which (i) increases the amount of tumor antigens,including neoantigens, which are released for the induction of ananti-tumor immune response; and (ii) enhances the expression of thevirus-encoded immune stimulatory molecule(s). Expression of immunestimulatory molecule(s) by the virus can further enhance and potentiatethe anti-tumor immune effect. Expression of fusogenic protein(s) by thevirus can further enhance viral spread through tumors. Expression offusogenic protein(s) by the virus can further enhance tumor cellkilling.

Anti-tumor efficacy of an oncolytic virus of the invention is achievedwhen the virus is used as a single agent and also when the virus is usedin combination with other anti-cancer modalities, includingchemotherapy, treatment with targeted agents, radiation, immunecheckpoint blockade (i.e. administration of one or more antagonist of animmune co-inhibitory pathway) and/or immune potentiating drugs (e.g.oneor more agonists of an immune co-stimulatory pathway). The improveddirect oncolytic effects (i.e. virus replication in, spread between, anddirect killing of tumor cells) and improved systemic anti-tumor immuneeffects of the viruses of the invention improve on the combined benefitsof oncolytic therapy and immune co-inhibitory pathway blockade and/orimmune co-stimulatory pathway activation.

Accordingly, the present invention provides an oncolytic virus which is,or is derived from, a clinical isolate which has been selected bycomparing the abilities of a panel of three or more clinical isolates ofthe same viral species to kill tumor cells of two or more tumor celllines in vitro and selecting a clinical isolate which is capable ofkilling cells of two or more tumor cell lines more rapidly and/or at alower dose in vitro than one or more of the other clinical isolates inthe panel. The clinical isolate may be modified. A modified clinicalisolate may have mutations, such as deletions in the viral genome and/ormay express one or more heterologous genes.

The virus may be a strain of any virus species which may be used for theoncolytic treatment of cancer, including strains of herpes virus, poxvirus, adenovirus, retrovirus, rhabdovirus, paramyxovirus or reovirus.The virus is preferably a herpes simplex virus (HSV), such as HSV1. TheHSV typically does not express functional ICP34.5 and/or functionalICP47 and/or expresses the US11 gene as an immediate early gene.

The virus may comprise (i) a fusogenic protein-encoding gene; and/or(ii) an immune stimulator) molecule or an immune stimulatorymolecule-encoding gene. The virus may encode more than one fusogenicprotein andlor more than one immune stimulatory molecule. The fusogenicprotein is preferably the glycoprotein from gibbon ape leukemia virus(GALV) and has the R transmembrane peptide mutated or removed (GALV-R-).The immune stimulatory molecule is preferably GM-CSF and/or an agonistof an immune co-stimulatory pathway including GITRL, 4-1-BBL, OX40L,ICOSL or CD40L, or a modified version in each case thereof, or a proteincapable of blocking signaling through CTLA-4, for example an antibody ora fragment thereof which binds CTLA-4.

The invention also provides:

-   -   a pharmaceutical composition comprising a virus of the invention        and a pharmaceutically acceptable carrier or diluent;    -   the virus of the invention fhr use in a method of treating the        human or animal body by therapy;    -   the virus of the invention for use in a method of treating        cancer, wherein the method optionally comprises administering a        further anti-cancer agent;    -   a product of manufacture comprising a virus of the invention in        a sterile vial, ampoule or syringe;    -   a method of treating cancer, which comprises administering a        therapeutically effective amount of a virus or a pharmaceutical        composition of the invention to a patient in need thereof,        wherein the method optionally comprises administering a further        anti-cancer agent;    -   use of a virus of the invention in the manufacture of a        medicament for use in a method of treating cancer, wherein the        method optionally comprises administering a further anti-cancer        agent, which is optionally an antagonist of an immune        co-inhibitory pathway, or an agonist of an immune co-stimulatory        pathway;    -   a method of treating cancer, which comprises administering a        therapeutically effective amount of an oncolytic virus, an        inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway and a        further antagonist of an immune co-inhibitory pathway, or        agonist of an immune co-stimulatory pathway to a patient in need        thereof; and    -   a method of selecting an oncolytic virus, the method comprising:        -   (i) comparing the abilities of a panel of three or more            clinical isolates of the same viral strain to kill tumor            cells of two or more tumor cell lines in vitro;        -   (ii) scoring the abilities of each of the panel of viruses            to kill tumor cells;        -   (iii) selecting a virus which has one of the best scores;        -   (iv) optionally modifying the virus to inactivate one or            more viral genes; and/or        -   (v) optionally modifying the virus to express one or more            immune stimulatory molecule encoding genes and/or one or            more fusogenic protein-encoding genes.

The further anti-cancer agent may be an antagonist of an immuneco-inhibitory pathway or an agonist of an immune co-stimulatory pathway.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the structure of an exemplary virus of the invention thatcomprises a gene encoding GALV-R- and a gene encoding GM-CSF insertedinto the ICP34.5 gene locus, and in which the ICP47 gene is deleted suchthat the US11 gene is under the control of the ICP47 immediate earlypromoter (top panel). FIG. 1 also shows similar exemplary viruses of theinvention expressing only a GALV-R-encoding gene (second panel), or onlya GM-CSF-encoding gene (third panel) Also shown is an exemplary virus inwhich the ICP34.5 gene and the ICP47 gene are deleted.

FIG. 2 depicts the structure of an exemplary virus of the invention thatcomprises a gene encoding GALV-R-, a gene encoding GM-CSF and a geneencoding CD40L.

FIG. 3 shows the differential abilities of the eight top ranking HSV1clinical isolate strains as assessed by crystal violet staining 24 hoursor 48 hours after infection with a MOI of 0.1, 0.01 or 0.001 asindicated in the Figure to kill Fadu, SK-mel-28, A549, HT1080,MIA-PA-CA-2, HT29 and MDA-MB-23I human tumor cell lines. The virusstrains ranked first and second on each cell line are indicated. Thevirus RH018A was ranked first on each of the Fadu, HT1080, MIA-PA-CA-2and HT29 cell lines and second on each of the SK-mel-2.8, A549 andMDA-MB-231 cell lines. RH004A was ranked joint first with RH018A andRH015A on the HT29 cell line, first on the SK-mel-28 and A549 cell linesand second on the Fadu cell line, RI-1023A was ranked first on theMDA-MB-231 cell line and second on the HT1080 cell line. RH031A wasranked second on each of the MIA-PA-CA-2 and HT29 cell lines. RH040A wasranked joint second on the HT29 cell line.

FIG. 4 shows a comparison between strain RH018A, the strain ranked firstof all the strains tested, with an ‘average’ strain from the screen(i,e. strain RH065A). Approximately 10 fold less of strain RH018A wasneeded to kill an equal proportion of cells than was needed of strainRH065A as shown by crystal violet staining 24 or 48 hours post infectionwith MOIs of 0.1, 0.01 and 0.001 in SK-mel-28, HT1080, MDA-MB-231, Fadu,MIA-PA-CA-2 and A549 cell lines.

FIG. 5 depicts structures of HSV1 viruses modified by the deletion ofICP34.5 and ICP47 such that the US11 gene is under control of the ICP457immediate early promoter and containing heterologous genes in theICP34.5 locus. The viruses were constructed using the RH018A strainunless otherwise stated in the Figure.

FIG. 6 shows the results of an ELISA to detect expression of human ormouse GM-CSF in supernatants from BHK cells infected with virus 16(mGM-CSF and GALVR-), virus 17 (hGM-CSF and GALVR-) and virus 19(mGM-CSF).

FIG. 7 is a comparison between the cell-killing abilities of strainRH018A in which ICP34.5 is deleted and which expresses GALVR- and GFP(virus 10) with a virus that expresses only GFP (virus 12) as determinedby crystal violet staining in three cell lines at low magnification.

FIG. 8 is a comparison between the cell-killing abilities of strainRH018A in which ICP34.5 and ICP47 are deleted and which expresses GALVR-and GM-CSF (virus 17) with a prior art strain with the samemodifications as determined by crystal violet staining in four celllines.

FIG. 9 shows the effectiveness of Virus 16 (ICP34.5 and ICP47 deletedexpressing GALVR- and mGM-CSF) in treating mice harbouring A20 lymphomatumors in both flanks. Tumors on the right flanks were injected with thevirus or vehicle and the effects on tumor size was observed for 30 days.The virus was effective against both injected tumors and non-injectedtumors.

FIG. 10 demonstrates the effects of Virus 15 (ICP34.5 and ICP47 deletedexpressing GALVR- and GFP) and Virus 24 (ICP34.5 and ICP47 deletedexpressing GFP) on rat 9L cells in vitro as assessed by crystal violetstaining. The virus expressing GALV (Virus 15) showed enhanced killingof rat 9L cells in vitro as compared to a virus which does not expressGALV (Virus 24).

FIG. 11 shows the antitumor effects of Virus 16 in Balb/c mice harboringmouse CT26 tumors in the left and right flanks. Groups of 10 mice werethen treated with: Vehicle (3 injections into right flank tumors everyother day); 5×10exp6 pfu of Virus 16 (mRP1) injected in the right flanktumor every other day; anti-mouse PD1 alone (10 mg/kg i.p. every threedays, BioXCell clone RMP1-14); anti-mouse CTLA-4 (3 mg/kg i.p everythree days, BioXCell clone 9D9); anti-mouse PD1 together with Virus 16;anti-mouse CTLA4 together with Virus 16; 1-methyl trypotophan (I-MT; IDOinhibitor (5 mg/ml in drinking water)); anti-mouse PD1 together with1-methyl trypotophan; or anti-mouse PD1 together with 1-methyltrypotophan and Virus 16. Effects on tumor size were observed for afurther 30 days. Greater tumor reduction was seen in animals treatedwith combinations of virus and checkpoint bockade than with the singletreatment groups. FIG. 11A shows that using Virus 16 and anti-PD1 incombination has a better anti-tumor effect than using either anti-PD1 orthe virus alone. FIG. 11B shows that the anti-tumor effect of Virus 16in combination with anti-CTLA-4 was better than the anti-tumor effect ofeither Virus 16 or anti-CTLA-4 alone. FIG. 11C shows that enhanced tumorreduction was observed using Virus 16 together with both anti-PD1 andIDO inhibition as compared to anti-PD1 and 1-MT inhibition in theabsence of the virus.

FIG. 12 shows the enhanced anti-tumor activity of Virus 16 incombination with immune checkpoint blockade in mouse A20 tumors in bothflanks of Balb/c mice as compared to either virus alone or checkpointblockade alone (anti-PD1).

FIG. 13 shows the structure of ICP34.5 and ICP47 deleted virusesexpressing GALVR-, GM-CSF and codon optimized anti-mouse or anti-humanCTLA-4 antibody constructs (secreted scFv molecules linked to human ormouse IgG1 Fc regions). The scFvs contain the linked ([G₄S]₃) light andheavy variable chains from antibody 9D9 (US2011044953: mouse version)and from ipilimumab (US20150283234; human version). The resultingstructure of the CTLA-4 inhibitor is also shown.

FIG. 14 shows anti-tumor effects of Virus 16 and Virus 19 in a humanxenograft model (A549). There were three injections of Virus 16, Virus19 or of vehicle over one week at three different dose levels(N=10/group). The doses of the viruses used is indicated. The anti-tumoreffects of Virus 16 which expresses GALV were better than those of Virus19 which does not express GALV.

FIG. 15 demonstrates the effects of viruses of the invention expressingGALVR- on 9L cells in the flanks of Fischer 344 rats. The followingtreatments were administered to groups of rats (ten per group), into oneflank of each rat only three times per week for three weeks: 50 μl ofvehicle; 50μl of 10⁷ pfu/ml of Virus 19 (expresses mGM-CSF but not GALVR-); or 50μl of 10⁷ pfu/ml of Virus 16 (expresses both mouse GM-CSF andGALV-R-). Effects on tumor growth were then observed for a further 30days. Superior tumor control and shrinkage was observed with the virusexpressing GM-CSF and GALV-R- as compared to the virus expressing GM-CSFalone.

FIG. 16 shows the anti-tumor effects of viruses expressing anti-mCTLA-4(virus 27), mCD40L (virus 32), mOX40L (virus 35), m4-2BBL (virus 33),each also with mGM-CSF and GALV-R- compared to virus 16 (expresses GALVand mGM-CSF).

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 is the nucleotide sequence of mouse GM-CSF.

SEQ ID NO: 2 is the nucleotide sequence of a codon optimized version ofmouse GM-CSF.

SEQ ID NO: 3 is the nucleotide sequence of human GM-CSF.

SEQ ID NO: 4 is the nucleotide sequence of a codon optimized version ofhuman GM-CSF.

SEQ ID NO: 5 is the amino acid sequence of mouse GM-CSF.

SEQ ID NO: 6 is the amino acid sequence of human GM-CSF.

SEQ ID NO: 7 is the nucleotide sequence of GALV-R-.

SEQ ID NO: 8 is the nucleotide sequence of a codon optimized version ofGALV-R- (the first three nucleotides are optional).

SEQ ID NO: 9 is the amino acid sequence of GALV-R-.

SEQ ID NO: 10 is the nucleotide sequence of a codon optimized version ofa human membrane bound version of CD40L.

SEQ ID NO: 11 is the amino acid sequence of a human membrane boundversion of CD40L.

SEQ ID NO: 12 is the nucleotide sequence of a codon optimized version ofa multimeric secreted version of human CD40L.

SEQ ID NO: 13 is the amino acid sequence of a multimeric secretedversion of human CD40L.

SEQ ID NO: 14 is the nucleotide sequence of a codon optimized version ofa multimeric secreted version of mouse CD40L.

SEQ ID NO: 15 is the amino acid sequence of a multimeric secretedversion of mouse CD40L.

SEQ ID NO: 16 is a codon optimized version of the nucleotide sequence ofwild-type human CD40L.

SEQ ID NO: 17 is the amino acid sequence of wild-type human CD40L.

SEQ ID NO: 18 is a codon optimized version of the nucleotide sequence ofwild-type mouse CD40L.

SEQ ID NO: 19 is the amino acid sequence of wild-type mouse CD40L.

SEQ ID NO: 20 is the nucleotide sequence of a codon optimized version ofmurine 4-1BBL.

SEQ ID NO: 21 is the nucleotide sequence of a codon optimized version ofhuman 4-1BBL.

SEQ ID NO: 22 is the nucleotide sequence of a codon optimized version ofsecreted mouse 4-1BBL.

SEQ ID NO: 23 is the nucleotide sequence of a codon optimized version ofhuman secreted 4-1BBL.

SEQ ID NO: 24 is the nucleotide sequence of a codon optimized version ofmurine GITRL.

SEQ ID NO: 25 is the nucleotide sequence of a codon optimized version ofhuman GITRL.

SEQ ID NO: 26 is the nucleotide sequence of a codon optimized version ofsecreted murine GITRL.

SEQ ID NO: 27 is the nucleotide sequence of a codon optimized version ofsecreted human GITRL.

SEQ ID NO: 28 is the nucleotide sequence of a codon optimized version ofmurine OX40L.

SEQ ID NO: 29 is the nucleotide sequence of a codon optimized version ofhuman OX40L.

SEQ ID NO: 30 is the nucleotide sequence of a codon optimized version ofsecreted murine OX40L.

SEQ ID NO: 31 is the nucleotide sequence of a codon optimized version ofsecreted human OX40L.

SEQ ID NO: 32 is the nucleotide sequence of a codon optimized version ofmurine ICOSL.

SEQ ID NO: 33 is the nucleotide sequence of a codon optimized version ofhuman ICOSL.

SEQ ID NO: 34 is the nucleotide sequence of a murine scFv CTLA-4antibody. The first six and last eight nucleotides are restriction sitesadded for cloning purposes.

SEQ ID NO: 35 is the nucleotide sequence of a murine scFv CTLA-4antibody. The first six and last eight nucleotides are restriction sitesadded for cloning purposes.

SEQ ID NO: 36 is the nucleotide sequence of the CMV promoter.

SEQ ID NO: 37 is the nucleotide sequence of the RSV promoter.

SEQ ID NO: 38 is the nucleotide sequence of BGH polyA.

SEQ ID NO: 39 is the nucleotide sequence of SV40 late polyA.

SEQ ID NO: 40 is the nucleotide sequence of the SV40 enhancer promoter.

SEQ ID NO: 41 is the nucleotide sequence of rabbit beta-globulin (RBG)polyA.

SEQ ID NO: 42 is the nucleotide sequence of GFP.

SEQ ID NO: 43 is the nucleotide sequence of the MoMuLV LTR promoter.

SEQ ID NO: 44 is the nucleotide sequence of the EF1a promoter.

SEQ ID NO: 45 is the nucleotide sequence of HGH polyA.

DETAILED DESCRIPTION OF THE INVENTION Oncolytic Virus

The virus of the invention is oncolytic. An oncolytic virus is a virusthat infects and replicates in tumor cells, such that the tumor cellsare killed. Therefore, the virus of the invention is replicationcompetent. Preferably, the virus is selectively replication competent intumor tissue. A virus is selectively replication competent in tumortissue if it replicates more effectively in tumor tissue than innon-tumor tissue. The ability of a virus to replicate in differenttissue types can be determined using standard techniques in the art.

The virus of the invention may be any virus which has these properties,including a herpes virus, pox virus, adenovirus, retrovirus,rhabdovirus, paramyxovirus or reovirus, or any species or strain withinthese larger groups. Viruses of the invention may be wild type (i.e.unaltered from the parental virus species), or with gene disruptions orgene additions. Which of these is the case will depend on the virusspecies to be used. Preferably the virus is a species of herpes virus,more preferably a strain of HSV, including strains of HSV1 and HSV2, andis most preferably a strain of HSV1. The virus of the invention is basedon a clinical isolate of the virus species to be used. The clinicalisolate is selected on the basis of it having particular advantageousproperties for the treatment of cancer. The virus of the invention hassurprisingly good anti-tumor effects compared to other strains of thesame virus isolated from other patients, wherein a patient is anindividual harbouring the virus species to be tested. The virus strainsused for comparison to identify viruses of the invention may be isolatedfrom a patient or an otherwise healthy (i.e. other than harboring thevirus species to be tested) volunteer, preferably an otherwise healthyvolunteer. HSV1 strains used to identify a virus of the invention aretypically isolated from cold sores of individuals harboring HSV1,typically by taking a swab using e.g. Virocult (Sigma) brandswab/container containing transport media followed by transport to thefacility to be used for further testing.

After isolation of viruses to be compared from individuals, stocks ofthe viruses are typically prepared, for example by growing the isolatedviruses on BHK or vero cells. Preferably, this is done following no morethan 3 cycles of freeze thaw between taking the sample and it beinggrown on, for example, BHK or vero cells to prepare the virus stock forfurther use. More preferably the virus sample has undergone 2 or lessthan 2 cycles of freeze thaw prior to preparation of the stock forfurther use, more preferably one cycle of freeze thaw, most preferablyno cycles of freeze thaw. Lysates from the cell lines infected with theviruses prepared in this way after isolation are compared, typically bytesting for the ability of the virus to kill tumor cell lines in vitro.Alternatively, the viral stocks may be stored under suitable conditions,for example by freezing, prior to testing. Viruses of the invention havesurprisingly good anti-tumor effects compared to other strains of thesame virus isolated from other individuals, preferably when compared tothose isolated from >5 individuals, more preferably >10 otherindividuals, most preferably >20 other individuals.

The stocks of the clinical isolates identified as viruses of theinvention (i.e. having surprisingly good properties for the killing oftumor cells as compared to other viral strains to which they werecompared) may be stored under suitable conditions, before or aftermodification, and used to generate further stocks as appropriate.

A clinical isolate is a strain of a virus species which has beenisolated from its natural host. The clinical isolate has preferably beenisolated for the purposes of testing and comparing the clinical isolatewith other clinical isolates of that virus species for a desiredproperty, in the case of viruses of the invention that being the abilityto kill human tumor cells. Clinical isolates which may be used forcomparison also include those from clinical samples present in clinicalrepositories, i,e. previously collected for clinical diagnostic or otherpurposes. In either case the clinical isolates used for comparison andidentification of viruses of the invention will preferably haveundergone minimal culture in vitro prior to being tested for the desiredproperty, preferably having only undergone sufficient culture to enablegeneration of sufficient stocks for comparative testing purposes. Assuch, the viruses used for comparison to identify viruses of theinvention may also include deposited strains, wherein the depositedstrain has been isolated from a patient, preferably an HSV1 strainisolated from the cold sore of a patient.

The virus of the invention is an oncolytic virus which is, or is derivedfrom, a clinical isolate which has been selected by comparing theabilities of a panel of three or more clinical isolates of the sameviral species to kill tumor cells of two or more tumor cell lines invitro and selecting a clinical isolate which is capable of killing cellsof two or more tumor cell lines more rapidly and/or at a lower dose invitro than one or more of the other clinical isolates in the panel.Thus, the virus is a clinical isolate that kills two or more tumor celllines more rapidly and/or at a lower dose in vitro than one or morereference clinical isolates of the same species of virus.

Typically, the clinical isolate of the invention will kill two or moretumor cell lines within 72 hours, preferably within 48 hours, morepreferably within 24 hours, of infection at multiplicities of infection.(MOI) of less than or equal to 0.1, preferably less than or equal to anMOI of 0.01 more preferably less than or equal to an MOI of 0.001.Preferably the clinical isolate will kill a broad range of human tumorcell lines, such as 2, 3, 4, 5, 6, 7 or all of the following cell lines:HT29 (colorectal), MDA-MB-231 (breast), SK-MEL-28 (melanoma), Fadu(squamous cell carcinoma), MCF7 (breast), A549 (lung), MIAPACA-2(pancreas), HT1080 (fibrosarcoma). Thus, the virus of the invention maybe capable of killing cells from two or more, such as 3, 4, 5, 6, 7 ormore, different types of tumor such as two or more, such as 3, 4, 5, 6,7 or more, solid tumors, including but not limited to colorectal tumorcells, prostate tumor cells, breast tumor cells, ovarian tumor cells,melanoma cells, squamous cell carcinoma cells, lung tumor cells,pancreatic tumor cells, sarcoma cells and/or fibrosarcoma cells.

Tumor cell line killing can be determined by any suitable method.Typically, a sample is first isolated from a patient, preferably, in thecase of HSV1, from a cold sore, is used to infect BHK cells, or anothersuitable cell line such as vero cells. Positive samples are typicallyidentified by the presence of a cytopathic effect (CPE) 24-72 hours postinfection, such as 48 hours post infection, and confirmed to be thetarget viral species by, for example, immunohistochemistry or PCR. Viralstocks are then generated from the positive samples. A sample from theviral stock is typically tested and compared to other samples generatedin the same way using swabs from different patients. Testing may becarried out by determining the level of CPE achieved at a range ofmultiplicity of infection (MOI) and at various times post infection.

For example, cell lines at 80% confluency may be infected with the viralsample at MOI of 1, 0.1, 0.01 and 0.001 and duplicate plates incubatedfor 24 and 48 hours at 37° C., 5% CO₂ prior to determination of theextent of viral cell killing. This may be determined by, for example,fixing the cells with glutaraldehyde and staining with crystal violetusing standard methods. The level of cell lysis may then be assessed bystandard methods such as gross observation, microscopy (cell counts) andphotography. The method may be repeated with the cells being incubatedfor shorter time periods, such as 8, 12 or 16 hours, or longer timeperiods, such as 72 hours, before cell killing is determined, or atadditional MOIs such as 0.0001 or less.

Growth curve experiments may also be conducted to assess the abilitiesof different clinical isolates to replicate in tumor cell lines invitro. For example, cell lines at 80% confluency may be infected withthe viral sample at MOI of 1, 0.1, 0.01 and 0.001 are incubated at 37°C., 5% CO₂ and the cells lysed, typically by freeze/thawing, at 0, 8,16, 24 and 48 hours post infection prior to determination of the extentof viral cell killing. This may be determined by, for example, assessingviral titres by a standard plaque assay.

A clinical isolate of the invention can kill infected tumor cell linesmore rapidly and/or at a lower MOI than the other clinical isolates towhich it is compared, preferably 2, 3, 4, 5 or 10 or more, otherclinical isolates of the same virus species. The clinical isolates ofthe invention typically kills a 10%, 25% or 50% greater proportion ofthe tumor cells present at a particular MOI and time point than at leastone, preferably 2, 3, 4, 5 or 10 or more, other clinical isolates of thesame virus type at the same MOI and time point to which it was compared.The clinical isolate of the invention typically kills the same or agreater proportion of tumor cells at a MOI that is half or less thanhalf that of the MOI at which one or more, preferably 2, 3, 4, 5,10 or15 or more, other clinical isolates of the same virus species used forthe comparison at the same time point, typically at 12, 24 and/or 48hours, kills the same proportion of tumor cells. Preferably, a clinicalisolate of the invention typically kills the same or a greaterproportion of tumor cells at a MOI that is 5 or 10 times lower than theMOI at which one or more, preferably 2, 3, 4, 5, 10 or 15 or more, otherclinical isolates of the same virus used for the comparison at the sametime point, typically at 12, 24 and/or 48 hours kills the sameproportion of tumor cells. The improved tumor cell killing abilities ofa virus of the invention are typically achieved compared to at least50%, 75% or 90% of the other clinical isolates of the same viral speciesused for the comparison. The virus is preferably compared to at least 4other virus strains, such as, for example, 7, 9, 19, 39 or 49 othervirus strains of the same species.

The isolated strains may be tested in batches, for example of 4-8 viralstrains at a time, on, for example, 4-8 of the tumor cell lines at atime. For each batch of experiments, the degree of killing achieved isranked on each cell line for the best (i.e. least surviving cells ateach time point/MOI) to the worst (i.e. most surviving cells for eachtime point/MOI) for the viruses being compared in that experiment. Thevirus strains from each experiment which perform the best across therange of tumor cell line tested (i.e. that consistently ranked as one ofthe best at killing the cell lines) may then be compared head to head infurther experiments using other clinical isolates and/ore other tumorcell lines to identify the best virus strains in the total of, forexample, >20 virus strains sampled. Those ranked as the best overall arethe viruses of the invention.

In a preferred embodiment, the virus of the invention is a strainselected from:

strain RH018A having the provisional accession number ECCAC 16121904;

strain RH004A having the provisional accession number ECCAC 16121902;

strain RH031A having the provisional accession number ECCAC 16121907;

strain RH040A having the provisional accession number ECCAC 16121908;

strain RH015A having the provisional accession number ECCAC 16121903;

strain RH021A having the provisional accession number ECCAC 16121905;

strain RH023A having the provisional accession number ECCAC 16121906;and

strain RH047A having the provisional accession number ECCAC 16121909.

More preferably, the virus of the invention is a strain selected from:

strain RH018A having the provisional accession number ECCAC 16121904;

strain RH004A having the provisional accession number ECCAC 16121902;

strain RH031A having the provisional accession number ECCAC 16121907;

strain RH040A having the provisional accession number ECCAC 16121908;and

strain RH015A having the provisional accession number ECCAC 16121903;

Most preferably, the virus of the invention is strain RH018A having theaccession number EACC 16121904.

An HSV of the invention is capable of replicating selectively in tumors,such as human tumors. Typically, the HSV replicates efficiently intarget tumors but does not replicate efficiently in non-tumor tissue.This HSV comprises one or more mutations in one or more viral genes thatinhibit replication in normal tissue but still allow replication intumors. The mutation may, for example, be a mutation that prevents theexpression of functional ICP34.5, ICP6 and/or thymidine kinase by theHSV.

In one preferred embodiment, the ICP34.5-encoding genes are mutated toconfer selective oncolytic activity on the HSV. Mutations of theICP34.5-encoding genes that prevent the expression of functional ICP34.5are described in Chou et al. (1990) Science 250:1262-1266, Maclean etal. (1991) J. Gen. Virol. 72:631-639 and Liu et al. (2003) Gene Therapy10:292-303, which are incorporated herein by reference. TheICP6-encoding gene and/or thymidine kinase-encoding gene may also beinactivated, as may other genes provided that such inactivation does notprevent the virus infecting or replicating in tumors.

The HSV may contain a further mutation or mutations which enhancereplication of the HSV in tumors. The resulting enhancement of viralreplication in tumors not only results in improved direct ‘oncolytic’tumor cell killing by the virus, but also enhances the level ofheterologous (i.e. a gene inserted into the virus, in the case ofviruses of the invention genes encoding fusogenic protein(s) and animmune modulatory molecule(s)) gene expression and increases the amountof tumor antigen released as tumor cells die, both of which may alsoimprove the immunogenic properties of the therapy for the treatment ofcancer. For example, in a preferred embodiment of the invention,deletion of the ICP47-encoding gene in a manner that places the US11gene under the control of the immediate early promoter that normallycontrols expression of the ICP47 encoding gene leads to enhancedreplication in tumors (see Liu et al., 2003, which is incorporatedherein by reference).

Other mutations that place the US11 coding sequence, which is an HSVlate gene, under the control of a promoter that is not dependent onviral replication may also be introduced into a virus of the invention.Such mutations allow expression of US11 before HSV replication occursand enhance viral replication in tumors. In particular, such mutationsenhance replication of an HSV lacking functional ICP34.5-encoding genes.

Accordingly, in one embodiment the HSV of the invention comprises a US11gene operably linked to a promoter, wherein the activity of the promoteris not dependent on viral replication. The promoter may be an immediateearly (IE) promoter or a non-HSV promoter which is active in mammalian,preferably human, tumor cells. The promoter may, for example, be aeukaryotic promoter, such as a promoter derived from the genome of amammal, preferably a human. The promoter may be a ubiquitous promoter(such as a promoter of (β-actin or tubulin) or a cell-specific promoter,such as tumor-specific promoter. The promoter may be a viral promoter,such as the Moloney murine leukaemia virus long terminal repeat (MMLVLTR) promoter or the human or mouse cytomegalovirus (CMV) IE promoter.HSV immediate early (IE) promoters are well known in the art. The HSV IEpromoter may be the promoter driving expression of ICP0, ICP4, ICP22,ICP27 or ICP47.

The genes referred to above may be rendered functionally inactive by anysuitable method, for example by deletion or substitution of all or partof the gene and/or control sequence of the gene or by insertion of oneor more nucleic acids into or in place of the gene and/or the controlsequence of the gene. For example, homologous recombination methods,which are standard in the art, may be used to generate the virus of theinvention.

As used herein, the term “gene” is intended to mean the nucleotidesequence encoding a protein, i.e. the coding sequence of the gene. Thevarious genes referred to above may be rendered non-functional bymutating the gene itself or the control sequences flanking the gene, forexample the promoter sequence. Deletions may remove one or more portionsof the gene, the entire gene or the entire gene and all or some of thecontrol sequences. For example, deletion of only one nucleotide withinthe gene may be made, resulting in a frame shift. However, a largerdeletion may be made, for example at least about 25%, more preferably atleast about 50% of the total coding and/or non-coding sequence. In onepreferred embodiment, the gene being rendered functionally inactive isdeleted. For example, the entire gene and optionally some of theflanking sequences may be removed from the virus. Where two or morecopies of the gene are present in the viral genome both copies of thegene are rendered functionally inactive.

A gene may be inactivated by substituting other sequences, for exampleby substituting all or part of the endogenous gene with a heterologousgene and optionally a promoter sequence. Where no promoter sequence issubstituted, the heterologous gene may be inserted such that it iscontrolled by the promoter of the gene being rendered non-functional. Inan HSV of the invention it is preferred that the ICP34.5 encoding-genesare rendered non-functional by the insertion of a heterologous gene orgenes and a promoter sequence or sequences operably linked thereto, andoptionally other regulatory elements such as polyadenylation sequences,into each the ICP34.5-encoding gene loci.

A virus of the invention may be used to express a fusogenic proteinand/or an immune stimulatory protein in tumors. This is typicallyachieved by inserting a heterologous gene encoding the fusogenic proteinand/or a heterologous gene encoding the immune stimulatory protein inthe genome of a selectively replication competent virus wherein eachgene is under the control of a promoter sequence. As replication of sucha virus will occur selectively in tumor tissue, expression of thefusogenic protein and/or immune stimulatory protein by the virus is alsoenhanced in tumor tissue as compared to non-tumor tissue in the body.Enhanced expression occurs where expression is greater in tumors ascompared to other tissues of the body. Accordingly, the inventionprovides benefits of expression of both a fusogenic protein and/or animmune stimulatory protein selectively in tumors combined with theanti-tumor effect provided by oncolytic virus replication.

Fusogenic Protein

The virus of the invention may comprise a gene encoding a fusogenicprotein. The fusogenic protein may be any heterologous protein capableof promoting fusion of a cell infected with the virus of the inventionto another cell. A fusogenic protein, preferably a wild type or modifiedviral glycoprotein (i.e. modified to increase its fusogenic properties),is a protein which is capable in inducing the cell to cell fusion(syncitia formation) of cells in which it is expressed. Examples offusogenic glycoprotiens include VSV-G, syncitin-1 (from human endogenousretrovirus-W (HERV-W)) or syncitin-2 (from HERVFRDE1), paramyxovirusSV5-F, measles virus-H, measles virus-F, RSV-F, the glycoprotein from aretrovirus or lentivirus, such as gibbon ape leukemia virus (GALV),murine leukemia virus (MLV), Mason-Pfizer monkey virus (MPMV) and equineinfectious anemia virus (EIAV) with the R transmembrane peptide removed(R-versions). In a preferred embodiment the fusogenic protein is fromGALV and has the R-peptide removed (GALV-R-).

The virus of the invention may comprise multiple copies of the fusogenicprotein-encoding gene, preferably 1 or 2 copies. The virus may comprisetwo or more different fusogenic proteins, including any of the fusogenicproteins listed above.

The fusogenic protein or proteins expressed by a virus of the inventionmay be identical to a naturally occurring protein, or may be a modifiedprotein.

The fusogenic protein-encoding gene (fusogenic gene) may have anaturally occurring nucleic acid sequence or a modified sequence. Thesequence of the fusogenic gene may, for example, be modified to increasethe fusogenic properties of the encoded protein, or to provide codonoptimisation and therefore increase the efficiency of expression of theencoded protein.

Immune Stimulatory Molecule

The virus of the invention may comprise one or more immune stimulatorymolecules and/or one or more genes encoding an immune stimulatorymolecule Immune stimulatory molecules include proteins which may aid inthe induction of an immune response, proteins which may relieveinhibitory signals to the induction or effectiveness of an immuneresponse and RNA molecules (e.g. shRNA, antisense RNA, RNAi or microRNA) which inhibit the expression of immune inhibitory molecules.

Examples of immune stimulatory molecules include IL-2, IL12, IL-15,IL-18, IL-21, IL-24, CD40 ligand, GITR ligand, 4-1-BB ligand, OX40ligand, ICOS ligand, flt3 ligand, type I interferons, includinginterferon alpha and interferon beta, interferon gamma, type IIIinterferon (IL-28, IL-29), other cytokines such as TNF alpha or GM-CSF,TGF beta or immune checkpoint antagonists Immune checkpoint antagonistsinclude antibodies, single chain antibodies andRNA1/siRNA/microRNA/antisense RNA knockdown approaches. Agonists ofimmune potentiating/co-stimulatory pathways include mutant or wild type,soluble, secreted and/or membrane bound ligands, and agonisticantibodies including single chain antibodies. With regard to thetargeting of immune co-inhibitory or immune co-stimulatory pathways,proteins or other molecules (agonistic or antagonistic depending on thecase) targeting CTLA-4 (antagonist), PD-1 (antagonist), PD-L1(antagonist), LAG-3 (antagonist), TIM-3 (antagonist), VISTA(antagonist), CSF1R (antagonist), IDO (antagonist), CEACAM1(antagonist), GITR (agonist), 4-1-BB (agonist), KIR (antagonist), SLAMF7(antagonist), OX40 (agonist), CD40 (agonist), ICOS (agonist) or CD47(antagonist) are particularly preferred. Viruses of the inventiontherefore preferably encode one or more of these molecules. Morepreferably viruses of the invention encode GM-CSF and/or a wild type ormodified version of CD40L, ICOSL, 4-1-BBL, GITRL or OX40L, mostpreferably GM-CSF.

The inhibitor of a co-inhibitory pathway may be a CTLA-4 inhibitor. TheCTLA-4 inhibitor is typically a molecule such as a peptide or proteinthat binds to CTLA-4 and reduces or blocks signaling through CTLA-4,such as by reducing activation by B7. By reducing CTLA-4 signalling, theinhibitor reduces or removes the block of immune stimulatory pathways byCTLA-4.

The CTLA-4 inhibitor is preferably an antibody or an antigen bindingfragment thereof. The term “antibody” as referred to herein includeswhole antibodies and any antigen binding fragment (i.e.,“antigen-binding portion”) or single chains thereof. An antibody refersto a glycoprotein comprising at least two heavy (H) chains and two light(kappa)(L) chains inter-connected by disulfide bonds, or an antigenbinding portion thereof. Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as VH) and a heavy chain constantregion. Each light chain is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. Thevariable regions of the heavy and light chains contain a binding domainthat interacts with an antigen. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). The constant regions of theantibodies may mediate the binding of the immunoglobulin to host tissuesor factors, including various cells of the immune system (e.g., effectorcells) and the first component (Clq) of the classical complement system.

The antibody is typically a monoclonal antibody. The antibody may be achimeric antibody. The antibody is preferably a humanised antibody andis more preferably a human antibody.

The term “antigen-binding fragment” of an antibody refers to one or morefragments of an antibody that retain the ability to specifically bind toCTLA-4. The antigen-binding fragment also retains the ability to inhibitCTLA-4 and hence to reduce or remove the CTLA-4 blockade of astimulatory immune response. Examples of suitable fragments include aFab fragment, a F(ab′)₂ fragment, a Fab′ fragment, a Fd fragment, a Fvfragment, a dAb fragment and an isolated complementarity determiningregion (CDR). Single chain antibodies such as scFv and heavy chainantibodies such as VHH and camel antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody. Ina preferred embodiment, the antibody is an scFv. Examples of suitablescFv molecules are disclosed in, for example, WO2007/123737 andWO2014/066532, which are incorporated herein by reference. The scFv maybe encoded by the nucleotide sequence shown in SEQ ID NO: 34 thenucleotide sequence shown in SEQ ID NO: 35.

Viruses of the invention may encode one or more immune stimulatorymolecules, preferably 1, 2, 3 or 4 immune stimulatory molecules, morepreferably 1 or 2 immune stimulatory molecules.

The sequence of the gene encoding the immune stimulatory molecule may becodon optimized so as to increase expression levels of the respectiveproteins in target cells as compared to if the unaltered sequence isused.

Modification of Virus Strains

Modified viruses of the invention are modified versions of such clinicalisolates identified as having advantageous properties for killing tumorcells as compared to other virus strains used for the comparison.Modified viruses of the invention are constructed using methods wellknown in the art. For example plasmids (for smaller viruses and singleand multiple genome component RNA viruses) or BACS (for larger DNAviruses including herpes viruses) encoding the viral genome to bepackaged, including any genes encoding fusogenic and/or immunestimulating molecules under appropriate regulatory control, can beconstructed by standard molecular biology techniques and transfectedinto permissive cells from which recombinant viruses can be recovered.

Alternatively, in a preferred embodiment plasmids containing DNA regionsflanking the intended site of insertion can be constructed, and thenco-transfected into permissive cells with viral genomic DNA such thathomologous recombination between the target insertion site flankingregions in the plasmid and the same regions in the parental clinicalisolate occur. Recombinant viruses can then be selected and purifiedthrough the loss or addition of a function inserted or deleted by theplasmid used for modification, e.g. insertion or deletion of a markergene such as GFP or lacZ from the parental virus at the intendedinsertion site. In a most preferred embodiment the insertion site is theICP34.5 locus of HSV, and therefore the plasmid used for manipulationcontains HSV sequences flanking this insertion site, between which arean expression cassette encoding a fusogenic protein and an immunestimulatory molecule. In this case, the parental clinical isolate maycontain a cassette encoding GFP in place of ICP34.5 and recombinantvirus plaques are selected through the loss of expression of GFP. In amost preferred embodiment the US11 gene of HSV is also expressed as anIE gene. This may be accomplished through deletion of the ICP47-encodingregion, or by other means.

Fusogenic protein encoding sequences and immune stimulatory moleculeencoding sequences may be inserted into the viral genome underappropriate regulatory control. This may be under the regulatory controlof natural promoters of the virus species of the invention used,depending on the species and insertion site, or preferably under thecontrol of heterologous promoters. Suitable heterologous promotersinclude mammalian promoters, such as the IEF2a promoter or the actinpromoter. More preferred are strong viral promoters such as the CMV IEpromoter, the RSV LTR, the MMLV LTR or promoters derived from SV40.Preferably each exogenous gene (i.e. encoding the fusogenic protein andimmune modulatory molecule) will be under separate promoter control, butmay also be expressed from a single RNA transcript, for example throughinsertion of an internal ribosome entry sites (IRES) between proteincoding sequences. RNA derived from each promoter is typically terminatedusing a polyadenylation sequence (e.g. mammalian sequences such as thebovine growth hormone (BGH) poly A sequence, synthetic polyadenylationsequences, or viral sequences such as the SV40 early or latepolyadenylation sequence).

The invention also provides a virus, such as a pox virus or a HSV,preferably HSV1, which expresses at least three heterologous genes,wherein each of the three heterologous genes is driven by a differentpromoter selected from the CMV promoter, the RSV promoter, the EF1apromoter, the SV40 promoter and a retroviral LTR promoter. The virusmay, for example, express four heterologous genes, wherein each of thefour heterologous genes is driven by a different promoter selected fromthe CMV promoter, the RSV promoter, the EF1a promoter, the SV40 promoterand a retroviral LTR promoter. The retroviral LTR is preferably fromMMLV (SEQ ID NO:43), also knowm as MoMuLV. The heterologous genes may beterminated by poly adenylation sequences. The poly adenylation sequencesmay be the same or different. Preferably each heterologous gene isterminated by a different poly adenylation sequence, which is preferablyselected from the BGH, SV40, HGH and RBG poly adenylation sequences.

The invention also provides a virus, such as a pox virus or a HSV,preferably HSV1, which expresses at least three heterologous genes,wherein each of the three heterologous genes is terminated by adifferent poly adenylation sequence selected from the BGH, SV40, HGH andRBG poly adenylation sequences. The virus may, for example, express fourheterologous genes terminated by each of the BGH, SV40, HGH and RBG polyadenylation sequences, respectively.

Pharmaceutical Compositions

The invention provides a pharmaceutical composition comprising a virusof the invention and a pharmaceutically acceptable carrier or diluent.Suitable carriers and diluents include isotonic saline solutions, forexample phosphate-buffered saline. The composition may further compriseother constituents such as sugars or proteins to improve properties suchas stability of the product. Alternatively a lyophilized formulation maybe used, which is reconstituted in a pharmaceutically acceptable carrieror diluent before use.

The choice of carrier, if required, is frequently a function of theroute of delivery of the composition. Within this invention,compositions may be formulated for any suitable route and means ofadministration. Pharmaceutically acceptable carriers or diluents arethose used in compositions suitable for intra-tumoral administration,intravenous/intraarterial administration, administration into the brainor administration into a body cavity (e.g. bladder, pleural cavity or byintraperitoneal administration). The composition may be administered inany suitable form, preferably as a liquid.

The present invention also provides a product of manufacture comprisinga virus of the invention in a sterile vial, ampoule or syringe.

Medical Uses/Methods of Treatment

The invention provides the virus of the invention for use in thetreatment of the human or animal body by therapy, particularly for usein a method of treating cancer. The cancer is typically in a mammal,preferably in a human. The virus kills infected tumour cells by virusmediated toxicity, including by lysis, necrosis or apoptosis, preferablyby lysis or necrosis. The virus of the invention also elicits a systemicanti-tumor immune response, augmented through the expression of theimmune stimulatory molecule, which also kills cancer cells.

The invention also provides a method of treating cancer, the methodcomprising administering a therapeutically effective amount of the virusof the invention to an individual in need thereof.

The invention additionally provides the use of the virus of theinvention in the manufacture of a medicament for treating cancer.

The virus of the invention is particularly useful in treating any solidtumor including any adenocarcinoma, carcinoma or sarcoma. For example,the virus of the invention is useful in treating head and neck,prostate, breast, ovarian, lung, liver, endometrial, bladder, gallbladder, pancreas, colon, kidney, stomach/gastric, esophageal, orcervical cancers, mesothelioma, melanoma or other skin cancer, lymphoma,glioma or other cancer of the nervous system, or sarcomas such as softtissue sarcoma.

The virus of the invention may be used to treat malignant tumors,including tumors that have metastasised from the site of the originaltumor. In this embodiment, the virus may be administered to the primarytumor or to one or more secondary tumors.

The virus of the invention may be administered in combination with othertherapeutic agents, including chemotherapy, targeted therapy,immunotherapy (including immune co-inhibitory pathway blockade or immuneco-stimulatory pathway activation) and/or in combination withradiotherapy and/or in combination with any combination of these. Thetherapeutic agent is preferably an anti-cancer agent.

The virus of the invention may be administered in combination with asecond virus, such as a second oncolytic virus.

For example, the therapeutic agent may comprise an immunogen (includinga recombinant or naturally occurring antigen, including such an antigenor combination of antigens delivered as DNA or RNA in which it/they areencoded), to further stimulate an immune response, such as a cellular orhumoral immune response, to tumor cells, particularly tumor neoantigens.The therapeutic agent may be an agent intended to increase or potentiatean immune response, such as a cytokine, an agent intended to inhibit animmune checkpoint pathway or stimulate an immune potentiating pathway oran agent which inhibits the activity of regulatory T cells (Tregs).

The therapeutic agent may be an agent known for use in an existingcancer therapeutic treatment. The therapeutic agent may be radiotherapyor a chemotherapeutic agent. The therapeutic agent may be selected fromcyclophosmamide, alkylating-like agents such as cisplatin or melphalan,plant alkaloids and terpenoids such as vincristine or paclitaxel(Taxol), antimetabolites such as 5-fluorouracil, topoisomeraseinhibitors type I or II such as camptothecin or doxorubicin, cytotoxicantibiotics such as actinomycin, anthracyclines such as epirubicin,glucocorticoids such as triamcinolone, inhibitors of protein, DNA and/orRNA synthesis such as methotrexate and dacarbaxine, histone deacetylase(HDAC) inhibitors, or any other chemotherapy agent.

The therapeutic agent may be one, or a combination of:immunotherapeutics or immunomodulators, such as TLR agonists; agentsthat down-regulate T-regulatory cells such as cyclophosphamide; oragents designed to block immune checkpoints or stimulate immunepotentiating pathways, including but not limited to monoclonalantibodies, such as a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1inhibitor, a LAG-3 inhibitor, a TIM-3 inhibitor, a VISTA inhibitor, aCSF1R inhibitor, an IDO inhibitor, a CEACAM1 inhibitor, a GITR agonist,a 4-1-BB agonist, a KIR inhibitor, a SLAMF7 inhibitor, an OX40 agonist,a CD40 agonist, an ICOS agonist or a CD47 inhibitor. In a preferredembodiment, the therapeutic agent is a CTLA-4 inhibitor such as ananti-CTLA-4 antibody, a PD1 inhibitor, such as an anti-PD-1 antibody ora PD-L1 inhibitor such as an anti-PD-L1 antibody. Such inhibitors,agonists and antibodies can be generated and tested by standard methodsknown in the art.

Immunotherapeutic agents may also include bi-specific antibodies, cellbased-therapies based on dendritic cells, NK cells or engineered T cellssuch CAR-T cells or T cells expressing engineered T cell receptorsImmunotherapeutic agents also include agents that target a specificgenetic mutation which occurs in tumors, agents intended to induceimmune responses to specific tumor antigens or combinations of tumorantigens, including neoantigens and/or agents intended to activate theSTING/cGAS pathway, TLR or other innate immune response and/orinflammatory pathway, including intra-tumoral agents.

For example, a virus of the invention may be used: in combination withdacarbazine, a BRAF inhibitor and or CTLA-4, PD1 or PD-L1 blockade totreat melanoma; in combination with taxol, doxorubicin, vinorelbine,cyclophosphamide and/or gemcitabine to treat breast cancer; incombination with 5-fluorouracil and optionally leucovorin, irinoteacanand/or oxaliplatin to treat colorectal cancer; in combination withtaxol, carboplatin, vinorelbine and/or gemcitabine, PD-1 or PD-L1blockade to treat lung cancer; in combination with cisplatin and/orradiotherapy to treat head and neck cancer.

The therapeutic agent may be an inhibitor of the idoleamine2,3-dioxygenase (IDO) pathway. Examples of IDO inhibitors includeepacadostat (INCB024360), 1-methyl-tryptophan, indoximod(1-methyl-D-tryptophan), GDC-0919 or F001287.

The mechanism of action of IDO in suppressing anti-tumor immuneresponses may also suppress immune responses generated followingoncolytic virus therapy. IDO expression is induced by toll like receptor(TLR) activation and interferon-γ both of which may result fromoncolytic virus infection. One embodiment of the use of oncolytic virustherapy for cancer treatment includes combination of an oncolytic virus,including a virus expressing an immune stimulating protein or proteinsand/or a fusogenic protein, with an inhibitor of the IDO pathway andoptionally one or more further antagonist of an immune co-inhibitorypathway and/or one or more agonist of an immune co-stimulatory pathway,including those targeting CTLA-4, PD-1 and/or PD-L1.

The invention also provides a method of treating cancer, which comprisesadministering a therapeutically effective amount of an oncolytic virus,an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway and afurther antagonist of an immune co-inhibitory pathway, and/or an agonistof an immune co-stimulatory pathway to a patient in need thereof. Theoncolytic virus is preferably a modified clinical isolate. The oncolyticvirus is preferably a pox virus, more preferably a HSV, such as a HSV1and/or a HSV rendered functionally inactive for ICP34.5 and/or ICP47.The oncolytic virus may express an immune stimulating molecule, such asGM-CSF, and/or a fusogenic protein, such as the GALV fusogenicglycoprotein with the R sequence mutated or deleted. The furtherantagonist of an immune co-inhibitory pathway is preferably anantagonist of CTLA-4, an antagonist of PD1 or an antagonist of PD-L1.For example, the further antagonist of an immune co-inhibitory pathwaymay be an inhibitor of the interaction between PD1 and PD-L1.

Where a therapeutic agent and/or radiotherapy is used in conjunctionwith a virus of the invention, administration of the virus and thetherapeutic agent and/or radiotherapy may be contemporaneous orseparated by time. The composition of the invention may be administeredbefore, together with or after the therapeutic agent or radiotherapy.The method of treating cancer may comprise multiple administrations ofthe virus of the invention and/or of the therapeutic agent and/orradiotherapy. A skilled practitioner will readily be able to determinesuitable courses of administration of the virus and the therapeuticagent.

In preferred embodiments, in the case of combination with one or moreantagonist of an immune co-inhibitory pathway, one or more agonist of animmune co-stimulatory pathway and/or other immune potentiating agents,the virus of the invention is administered once or multiple times priorto the concurrent administration of the antagonist of an immuneco-inhibitory pathway, agonist of an immune co-stimulatory pathwayand/or other immune potentiating agent or agents thereafter, orconcurrent with the administration of the antagonist of an immuneco-inhibitory pathway, agonist of an immune co-stimulatory pathwayand/or other immune potentiating agent or agents without prioradministration of the virus of the invention.

The virus of the invention may be administered to a subject by anysuitable route. Typically, a virus of the invention is administered bydirect intra-tumoral injection, including through the use of imagingguidance to target the tumor or tumors. The virus may be administeredinto a body cavity, for example into the pleural cavity, bladder or byintra-peritoneal administration. The virus may be injected into a bloodvessel, preferably a blood vessel supplying a tumor.

Therapeutic agents which may be combined with a virus of the inventioncan be administered to a human or animal subject in vivo using a varietyof known routes and techniques. For example, the composition may beprovided as an injectable solution, suspension or emulsion andadministered via parenteral, subcutaneous, oral, epidermal, intradermal,intramuscular, interarterial, intraperitoneal, intravenous injectionusing a conventional needle and syringe, or using a liquid jet injectionsystem. The composition may be administered topically to skin or mucosaltissue, such as nasally, intratrachealy, intestinally, sublingually,rectally or vaginally, or provided as a finely divided spray suitablefor respiratory or pulmonary administration. In preferred embodiments,the compositions are administered by intravenous infusion, orally, ordirectly into a tumor.

The virus and/or therapeutic agent may be administered to a subject inan amount that is compatible with the dosage composition that will betherapeutically effective. The administration of the virus of theinvention is for a “therapeutic” purpose. As used herein, the term“therapeutic” or “treatment” includes any one or more of the followingas its objective: the prevention of any metastasis or further metastasisoccurring; the reduction or elimination of symptoms; the reduction orcomplete elimination of a tumor or cancer, an increase in the time toprogression of the patient's cancer; an increase in time to relapsefollowing treatment; or an increase in survival time.

Therapeutic treatment may be given to Stage I, II, III, or IV cancers,preferably Stage II, III or IV, more preferably Stage III or IV, pre- orpost-surgical intervention, preferably before surgical intervention(either for resection of primary or recurrent/metastatic disease), i.e.while residual tumor remains.

Therapeutic treatment may be carried out following direct injection ofthe virus composition into target tissue which may be the tumor, into abody cavity, or a blood vessel. As a guide, the amount of virusadministered is in the case of HSV in the range of from 10⁴ to 10¹⁰ pfu,preferably from 10⁵ to 10⁹ pfu. In the case of HSV, an initial lowerdose (e.g. 10⁴ to 10⁷ pfu) may be given to patients to seroconvertpatients who are seronegative for HSV and boost immunity in those whoare seropositive, followed by a higher dose then being given thereafter(e.g. 10⁶ to 10⁹ pfu). Typically up to 20 ml of a pharmaceuticalcomposition consisting essentially of the virus and a pharmaceuticallyacceptable suitable carrier or diluent may be used for direct injectioninto tumors, or up to 50 ml for administration into a body cavity (whichmay be subject to further dilution into an appropriate diluent beforeadministration) or into the bloodstream. However for some oncolytictherapy applications larger or smaller volumes may also be used,depending on the tumor and the administration route and site.

The routes of administration and dosages described are intended only asa guide since a skilled practitioner will be able to determine readilythe optimum route of administration and dosage. The dosage may bedetermined according to various parameters, especially according to thelocation of the tumor, the size of the tumor, the age, weight andcondition of the patient to be treated and the route of administration.Preferably the virus is administered by direct injection into the tumor.The virus may also be administered by injection into a blood vessel orinto a body cavity. The optimum route of administration will depend onthe location and size of the tumor. Multiple doses may be required toachieve an immunological or clinical effect, which, if required, will betypically administered between 2 days to 12 weeks apart, preferably3-days to 3 weeks apart. Repeat doses up to 5 years or more may begiven, preferably for up to one month to two years dependent on thespeed of response of the tumor type being treated and the response of aparticular patient, and any combination therapy which may also be beinggiven.

The following Examples illustrate the invention.

EXAMPLE 1 Clinical Isolates with Improved Anti-Tumor Effects

The virus species used to exemplify the invention is HSV, specificallyHSV1. Cold sore swabs were taken from more than 20 otherwise healthyvolunteers. A sample of each swab was used to infect BHK cells. Samplescontaining HSV1 were identified by the presence of a cytopathic effect(CPE) 24-72 hours post infection and by immunohistochemistry and viralstocks of the primary clinical isolates were generated from the positivesamples.

The abilities of the primary clinical isolates of HSV1 to kill a panelof human tumor-derived cell lines is tested and the virus strain withthe greatest ability to kill a broad range of these rapidly, and at lowdose is chosen. Tumor cell lines used for this comparison are HT29(colorectal), MDA-MB-231 (breast), SK-MEL-28 (melanoma), Fadu (squamouscell carcinoma), MCF7 (breast), A549 (lung), MIAPACA-2 (pancreas),CAPAN-1(pancreas), HT1080 (fibrosarcoma). The cell lines are used totest for the level of CPE achieved at a range of MOI and times postinfection for each of the primary clinical isolates.

More specifically, the tumor cell lines are used to seed multi-welltissue culture plates so that they are about 80% confluent on the day ofinfection. Representative wells from each tumor cell line aretrypsinised and the number of cells in the well determined. These cellcounts are used to determine the volume of each clinical isolaterequired to give an MOI of 1, 0.1, 0.01 and 0.001. Separate wells of atumor cell line are infected with the clinical isolate at these MOI andoverlaid with growth media and carboxymethylcellulose. All infectionsare carried out in quadruplicate. Duplicate wells are incubated for 24hours and duplicate wells are incubated for 48 hours, both at 37° C., 5%CO₂, prior to fixation of the cells with glutaraldehyde and stainingwith crystal violet. The level of cell lysis is then assessed by grossobservation, microscopy (cell counts) and photography or using ametabolic assay such as an MTT assay.

Growth curve experiments are also conducted to assess the abilities ofdifferent clinical isolates to replicate in tumor cell lines in vitro.The tumor cell lines are used to seed multi-well tissue culture platesso that they are about 80% confluent on the day of infection. Cellcounts are determined as above and used to determine the volume of virusto give MOIs of 1, 0.1, 0.01 and 0.001. The tumor cells are infected induplicate for MOI and time point. The infected cells are incubated at37° C., 5% CO₂ and the cells lysed by freeze/thawing at 0, 8, 16, 24 and48 hours post infection. Viral titres are assessed by a standard plaqueassay.

EXAMPLE 2 Modification of Clinical Isolates

In this example the clinical isolate selected in Example 1 (i.e. a virusif the invention) is modified by deletion of ICP47 from the viral genomeusing homologous recombination with a plasmid containing regionsflanking DISV1 nucleotides 145300 to 145582 (HSV1 nucleotides 145300 to145582 being the sequences to be deleted; HSV1 strain 17 sequenceGenbank file NC_001806.2) between which are encoded GFP. GFP expressingvirus plaques are selected, and GFP then removed by homologousrecombination with the empty flanking regions and plaques which do notexpress GEP are selected. This results in an ICP47 deleted virus inwhich US11 is expressed as an IE protein as it is now under the controlof the ICP47 promoter. ICP34.5 is then deleted using homologousrecombination with a plasmid containing regions flanking HSV1nucleotides 124953 to 125727 (HSV1 nucleotides 124953 to 125727 beingthe sequences to be deleted; HSV1 strain 17 sequence Genbank fileNC_001806.2) between which GFP is encoded. GFP expressing virus plaquesare again selected, and GFP then removed by homologous recombinationwith the same flanking regions but between which are now an expressioncassette comprising a codon optimized version of the mouse GM-CSFsequence and a codon optimized version of the GALV R- sequence driven bythe CMV IE promoter and RSV promoter respectively, in a back to backorientation and again selecting virus plaques which do not express GFP.This virus construction is performed using methods which are standard inthe art.

The structure of the resulting virus is shown in FIG. 1 (top panel). ThemGM-CSF and GALV-R- sequences are shown in SEQ ID NOs 2 and 8respectively. The structure of the resulting virus is confirmed byrestriction digestion and Southern blot, GM-CSF expression is confirmedby ELISA, and GALV-R- expression is confirmed by infection of humanHT1080 tumor cells and the observation of syncitial plaques.

Viruses are also constructed using similar procedures which have noinsertion into ICP34.5, or which only have inserted the gene for mouseGM-CSF or GALV-R-. The structures of these viruses are also shown inFIG. 1.

For human use, hGM-CSF is used, the sequence for a codon optimisedversion of which is shown in SEQ ID NO 4.

EXAMPLE 3 Expression of Two Immune Stimulatory Molecule from a VirusExpressing a Fusogenic Protein

A virus similar to the GALV-R- and mCGM-CSF expressing virus describedabove is constructed, but additionally expressing versions of CD40L.Here, instead of using a plasmid containing ICP34.5 flanking regions andan expression cassette comprising GM-CSF and GALV-R- driven by a CMV andan RSV promoter, a plasmid containing ICP34.5 flanking regions and anexpression cassette comprising GM-CSF, GALV and CD40L driven by a CMV,an RSV and an SV40 promoter is used for recombination with the viruscontaining GFP inserted into ICP34.5 and non-GFP expressing plaquesagain selected.

EXAMPLE 4 The Effect of the Combined Expression of a Fusogenic Proteinand an Immune Stimulatory Molecule from an Oncolytic Virus in MouseTumor Models

The GALV R- protein causes cell to cell fusion in human cells but not inmouse cells because the PiT-1 receptor required for cell fusion to occurhas a sequence in mice which does not allow cell fusion to occur. As aresult mouse tumor cells expressing human PiT-1 are first prepared usingmethods standard in the art. Human PiT-1 is cloned into a lentiviralvector also comprising a selectable marker gene. The vector istransfected into target CT26 mouse colorectal cancer tumor cells andclones resistant to the selectable marker are selected to generateCT26/PiT-1 cells, PiT-1 expression is confirmed by western blotting inuntransfected cells and in cells transfected with the PiT-1 expressinglentivirus and by transfection of a plasmid expressing GALV-R- andconfirmation that cell fusion occurs.

The utility of the invention is demonstrated by administering CT26/PiT-1cells into both flanks of Balb/c mice and allowing the CT26/PiT-1 tumorsto grow to approximately 0.5 cm in diameter.

The following treatments are then administered to groups of mice (fiveper group), into one flank of each mouse only 3 times per week for twoweeks:

-   -   50 μl of saline (1 group);    -   50 μl of 10⁵ pfu/ml, 10⁶ pfu, or 10⁷ pfu/ml of the HSV with no        inserted gene (3 groups);    -   50 μl of 10⁵ pfu/ml, 10⁶ pfu/ml, or 10⁷ pfu/ml of the HSV with        only mouse GM-CSF inserted (3 groups);    -   50 μl of 10⁵ pfu/ml, 10⁶ pfu/ml, or 10⁷ pfu/ml of the virus with        only GALV-R- inserted (3 groups); or    -   50 μl of 10⁵ pfu/ml, 10⁷ pfu/ml, or 10⁷ pfu/ml of the virus with        both mouse GM-CSF and GALV-R- inserted (3 groups).

Effects on tumor growth are then observed for up to one month. Superiortumor control and shrinkage in both injected and uninjected tumors withthe virus expressing GM-CSF and GALV-R- as compared to the other groupsis observed, including through an improved dose response curve.

EXAMPLE 5 The Effect of Combined Expression of a Fusogenic Protein andan Immune Stimulatory Molecule from an Oncolytic Virus on theTherapeutic Effect of Immune Checkpoint Blockade in Mouse Tumor Models

The experiment in Example 3 above is repeated but mice are additionallydosed bi-weekly by the intra-peritoneal route with an antibody targetingmouse PD-1 (10 mg/kg; Bioxcell RMP-1-14 on the same days as virusdosing) or an antibody targeting mouse CTLA-4 (10 mg/kg; Bioxcell 9H10on the same days as virus dosing). An additional group of mice is addedwhich receive no antibody treatment. More specifically, groups of micereceive (1) saline, (2) HSV with no inserted gene, (3) HSV with bothGM-CSF and GALV-R-inserted as in Example 3, (4) PD-1 antibody, (5)CTLA-4 antibody, (6) HSV with no inserted gene plus PD-1 antibody, (7)HSV with no inserted gene plus CTLA-4 antibody, (8) HSV with GM-CST andGALV-R- and PD-1 antibody or (9) HSV with GM-CSF and GALV-R- and CTLA-4antibody. Superior tumor control and shrinkage in both injected anduninjected tumors with the virus expressing GM-CSF and GAL,V-R- togetherwith the anti-PD-1 antibody or the anti-CTLA-4 antibody as compared tothe other groups is observed, including through an improved doseresponse curve.

EXAMPLE 6 Collection of Clinical Isolates

The virus species used to exemplify the invention is HSV, specificallyHSV1. To exemplify the invention, 181 volunteers were recruited whosuffered from recurrent cold sores. These volunteers were given samplecollection kits (including Sigma Virovult collection tubes), and usedthese to swab cold sores when they appeared following which thesesamples were shipped to Replimune, Oxford UK. From June 2015-February2016, swabs were received from 72 volunteers. A sample of each swab wasused to infect BHK cells. Of these 36 live virus samples were recoveredfollowing plating out and growth on BHK cells. These samples aredetailed in Table 1.

TABLE 1 Details of Tested Swab Samples & Result Sample Number Virusretrieved RH001A No RH001B RH002A Yes RH003A No RH004A Yes RH004B RH005ANo RH005B RH006A No RH006B RH007A Yes RH007B RH007C RH008A No RH008BRH008C RH009A No RH009B RH010A No RH011A No RH011B RH011C RH012A NoRH013A No RH014A Yes RH014B RH015A Yes RH016A No RH016B RH017A YesRH018A Yes RH018B RH018C RH019A No RH019B RH019C RH020A Yes- RH020A onlyRH020B RH020C RH021A Yes RH021B RH022A Yes RH022B RH023A Yes RH024A NoRH025A Yes -RH025B only RH025B RH026A Yes RH027A No RH027B RH027C RH028ANo RH028B RH028C RH029A No RH030A No RH031A Yes - RH031A to RH031BRH031D RH031C RH031D RH031E RH031F RH032A No RH033A No RH033B RH033CRH034A No RH034B RH034C RH035A No RH036A Yes RH037A Yes RH038A YesRH039A No RH039B RH039C RH040A Yes RH040B RH040C RH041A Yes RH042A YesRH043A No RH043B RH043C RH044A No RH045A No RH046A Yes RH047A Yes-RH047A and RH047B RH047C RH047C RH048A No RH049A No RH049B RH049C RH050ANo RH051A Yes RH051B RH052A Yes - RH052A only RH052B RH053A No RH054A NoRH055A No RH055B RH056A Yes RH057A No RH058A Yes RH058B RH059A No RH060ANo RH061A Yes RH062A No RH063A No RH064A Yes RH065A Yes RH065B RH066A NoRH067A No RH067B RH068A No - contaminated RH069A No RH069A RH070A YesRH071A Yes RH072A No RH073A Yes RH073B RH074A No RH074B RH075A No RH076ANo RH078A No RH078B RH079B Yes RH079B RH080A No RH081A Yes RH082A NoRH082B RH083A Yes RH083B RH084A Yes RH084B RH084C RH085A No RH086A NoRH087A Yes - RH078B only RH087BDesignations A, B, C etc. indicate multiple swabs from the samevolunteer.

EXAMPLE 7 Identification of Clinical Isolates with Improved Anti-TumorEffects

The abilities of the primary clinical isolates of HSV1 to kill a panelof human tumor-derived cell lines was tested. The tumor cell lines usedfor this comparison were HT29 (colorectal), MDA-MB-231 (breast),SK-MEL-28 (melanoma), Fadu (squamous cell carcinoma), MCF7 (breast),A549 (lung), MIAPACA-2 (pancreas) and HT1080 (fibrosarcoma). The celllines were used to test for the level of CPE achieved at a range of MOIand times post infection for each of the primary clinical isolates.

Experiments were conducted in parallel using 5 to 8 of the new virusesstrains at the same time. The virus strains were plated out in duplicateat a range of MOIs (0.001-1), and the extent of CPE following crystalviolet staining was assessed at 24 and 48 hours following infection. Theviral strains which were most effective at killing the tumor cell lineswere scored, and the most effective two or three strains from eachscreen of 5-8 strains were identified and compared in parallel in afurther experiment to identify the top strains for further development.

The initial screens demonstrated substantial variability in the abilityof the different strains to kill the different tumor cell lines. Of aninitial 29 strains tested, 8 strains of interest were identified in theinitial screens for further comparison. These were strains RH004A,RH015A, RH018A, RH021A, RH023A, RH31A, RH040A, and RH047A.

The 8 strains for further comparison were tested in parallel on thepanel of tumor cell lines, and their relative ability to kill thesetumor cell lines was assessed following crystal violet staining andobservation tbr CPE. FIG. 3 shows a representative time point and MOIfor these viruses on each of the viruses on each of the cell linesdemonstrating the differential ability of the viruses to kill the targettumor cell lines observed.

There was substantial variation amongst the strains, and it was foundthat while a particular strain may be particularly effective at killingone cell line, it is not necessarily particularly effective at killingother cell lines too, further demonstrating the degree of variability inthe ability of clinical strains of HSV to kill tumor cells of differenttypes.

FIG. 3 also indicates which of the virus strains was both best andsecond best at killing each of the cell lines, enabling the virusstrains to be rank ordered as to their overall relative ability to killthe panel of cell lines as a whole. This analysis demonstrated thatstrains RH004A, RH015A, RH018A, RH031A and RH040A were relatively moreeffective than the other strains, and these five strains were chosen forpotential further development as oncolytic agents. Of these top fivestrains, the relative rank order based on their abilities to kill acrossthe panel of cell lines was RH018A>RH004A>RH031A>RH040A>RH015A.

More specifically, in these experiments, the tumor cell lines were usedto seed multi-well tissue culture plates so that they were about 80%confluent on the day of infection. Representative wells from each tumorcell line were trypsinised and the number of cells in the welldetermined. These cell counts are used to determine the volume of eachclinical isolate required to give an MOI of 1, 0.1, 0.01 and 0.001.Separate wells of a tumor cell line were infected with the clinicalisolate at these MOI. All infections are carried out in quadruplicate.Duplicate wells were incubated for 24 hours and duplicate wells wereincubated for 48 hours, both at 37° C., 5% CO₂, prior to fixation of thecells with glutaraldehyde and staining with crystal violet. The level ofcell lysis was then assessed by gross observation, microscopy (cellcounts) and photography.

Strain RH018A, the strain ranked first of all the strains tested wascompared to an ‘average’ strain from the screen (i.e. a strain which wasnot in the top 8, but was also not in the group of strains which wereleast effective and killing the panel of tumor cell lines). Thiscomparison showed that Strain RH018A was approximately 10 fold moreeffective than this average strain (Strain RH065A) at killing the tumorcell lines (i.e. approximately 10 fold less of Strain RH018A was neededto kill an equal proportion of cells than was needed of Strain RH065A).This is shown in FIG. 4.

EXAMPLE 8 Modification of Clinical Isolates

In this Example the clinical isolates selected in Example 7 weremodified by deletion of ICP34.5 from the viral genome using homologousrecombination with a plasmid containing regions flanking the ICP34.5encoding gene (nucleotides 143680-145300 and 145,582-147,083 ; HSV1strain 17 sequence Genbank file NC_001806.2) between which are encodedGFP and the GALV-R-fusogenic glycoprotein. The structure of this virus,(Virus 10) is shown in FIG. 5.

Additional viruses based on Strain RH018A were also constructed in whichboth ICP34.5 and ICP47 (using flanking regions containing nucleotides123464-124953 and 125727-126781; HSV1 strain 17 sequence Genbank fileNC_001806.2) were deleted (resulting in placement of US11 under thecontrol of the ICP47 promoter). To construct these viruses, GFPexpressing virus plaques, with GFP expressed in place of IC47 were firstselected. GFP was then removed by homologous recombination with theempty flanking regions, and plaques not expressing GFP were selected.This resulted in an ICP47 deleted virus in which US11 is expressed as anIE protein as it is now under the control of the ICP47 promoter. ICP34.5was then deleted using homologous recombination with a plasmidcontaining regions flanking HSV1 nucleotides 143680-145300 and145,582-147,083; HSV1 strain 17 sequence Genbank tile NC_001806.2)between which GFP is encoded. GFP expressing virus plaques were againselected, and GFP then removed by homologous recombination with the sameflanking regions but between which are now an expression cassettecomprising the genes to be inserted. The viruses that were constructedare shown in FIGS. 1 and 5. These included a codon optimized version ofthe mouse GM-CSF sequence and a codon optimized version of the GALV R-sequence driven by the CMV IE promoter and RSV promoter respectively, ina back to back orientation and again selecting virus plaques which donot express GFP. This virus construction was performed using methodswhich are standard in the art.

The mGM-CSF and GALV-R- sequences are shown in SEQ ID NOs 2 and 8respectively. The structure of the resulting virus was confirmed by PCR,GM-CSF expression was confirmed by ELISA, and GALV-R- expression wasconfirmed by infection of human HT1080 tumor cells and the observationof syncitial plaques.

For human use, hGM-CSF is used, the sequence for a codon optimisedversion of which is shown in SEQ ID NO 4. The structure of this virus isshown in FIG. 5. Expression of mouse or human GM-CSF from viruses 16, 17and 19 is shown in FIG. 6.

EXAMPLE 9 A Virus of the in Modified for Oneolytic Use and Expressing aFusogenic Glycoprotein Shows Enhanced Tumor Cell Killing In Vitro asCompared to a Virus Which Does Not Express a Fusogenic Glycoprotein

Virus 10 (see FIG. 5), based on clinical Strain RH018A in which ICP34.5is deleted and which expresses GALVR- and GFP, was compared in vitro toa virus which expresses only GFP (Virus 12). Virus 10 showed enhancedkilling on a panel of human tumor cell lines as compared to Virus 12, asshown in FIG. 7.

EXAMPLE 10 A Virus of the Invention Modified for Oncolytic Use ShowsEnhanced Tumor Cell Killing as Compared to a Similarly Modified VirusWhich is Not of the Invention

Virus 17 (see FIG. 5), based on clinical Strain RH018A in which ICP34.5and ICP47 are deleted and which expresses GALVR- and GM-CSF, wascompared in vitro to a known virus which was also deleted for ICP34.5and ICP47 but which was not derived from a strain of the invention andwhich expresses only GM-CSF. Virus 17 showed enhanced killing on a panelof human tumor cell lines as compared to the previous virus, as shown inFIG. 8.

EXAMPLE 11 A Virus of the Invention Modified for Oncolytic UseEffectively Treats Mouse Tumors In Vivo

Virus 16 was tested in mice harboring A20 lymphoma tumors in the leftand right flanks. One million tumor cells were first implanted in bothflanks of Balb/c mice and tumors allowed to grow to 0.5-0.7 cm indiameter. Tumors on the right flank were then injected 3 times (everyother day) with either vehicle (10 mice) or 5×10exp6 pfu of Virus 16 (10mice), and effects on tumor size observed for a further 30 days. Thisdemonstrated that both injected and uninjected tumors were effectivelytreated with Virus 16 (see FIG. 9).

EXAMPLE 12 The Effect of the Combined Expression of a Fusogenic Proteinand an Immune Stimulatory Molecule from an Oneolytic Virus of theInvention in a Rat Tumor Model

The GALV R- protein causes cell to cell fusion in human cells but not inmouse cells. However, GALV R- does cause fusion in rat cells.

The utility of the invention was further demonstrated by administering9L cells into the flanks of Fischer 344 rats and allowing the 9L tumorsto grow to approximately 0.5 cm in diameter.

The following treatments were then administered to groups of rats (tenper group), into one flank only of each rat three times per week forthree weeks:

-   -   50 μl of vehicle;    -   50 μl of 10⁷ pfu/ml of Virus 19 (expresses mGM-CSF but not GALV        R-);    -   50 μl of 10⁷ pfu/ml of Virus 16 (expresses both mouse GM-CSF and        GALV-R-).

Effects on tumor growth were then observed for a further ≈30 days. Thisdemonstrated superior tumor control and shrinkage with the virusexpressing GALV-R in both injected and uninjected tumors, demonstratingimproved systemic effects. This is shown in FIG. 15. FIG. 10 shows thata virus expressing GALV (Virus 15) also shows enhanced killing of rat 91cells in vitro as compared to a virus which does not express GALV (Virus24).

EXAMPLE 13 A Virus of the Invention Modified for Oncolytic Use isSynergistic with Immune Checkpoint Blockade in Mouse Tumor Models

Virus 16 was tested in mice harboring CT26 tumors in the left and rightflanks. One million tumor cells were first implanted in both flanks ofBalb/c mice and tumors allowed to grow to 0.5-0.6 cm in diameter.

Groups of 10 mice were then treated with:

Vehicle (3 injections into right flank tumors every other day);

5×10exp6 pfu of Virus 16 injected in the right flank tumor every otherday;

anti-mousePD1 alone (10 mg/kg i.p. every three days, BioXCell cloneRMP1-14);

anti-mouseCTLA-4 (3 mg/Kg i.p every three days, BioXCell clone 9D9);

anti-mousePD1 together with Virus 16;

anti-mouseCLTA4 together with Virus 16;

1-methyl trypotophan (IDO inhibitor (5 mg/ml in drinking water));

anti-mouse P1)1 together with 1-methyl trypotophan;

anti-mouse PD1 together with 1-methyl trypotophan and Virus 16;

Effects on tumor size were observed for a further 30 days. A greatertumor reduction in animals treated with combinations of virus andcheckpoint blockade was demonstrated than in animals treated with thesingle treatment groups (see FIG. 11). Enhanced tumor reduction withVirus 16 together with both anti-PD1 and IDO inhibition was alsodemonstrated as compared to Virus 16 together with only anti-PD1 (seeFIG. 11).

Enhanced activity of Virus 16 in combination with immune checkpointblockade was also seen in A20 tumors (FIG. 12).

EXAMPLE 14 The Effect of the Expression of a Fusogenic Protein from anOncolytic Virus of the Invention in Human Xenograft Models in ImmuneDeficient Mice

The GALV R- protein causes cell to cell fusion in human cells but not inmouse cells. However, human xenograft tumors grown in immune deficientmice can be used to assess the effects of GALV expression on anti-tumorefficacy.

The utility of the invention was therefore further demonstrated byadministering A549 human lung cancer cells into the flanks of nude miceand allowing the tumors to grow to approximately 0.5 cm in diameter.

The following treatments were then administered to groups of mice (tenper group), into tumor containing flank of each mouse three times overone week:

-   -   50 μl of vehicle;    -   50 μl of 10⁷ pfu/ml of Virus 16 (expresses both mouse GM-CSF and        GALV-R-);    -   50 μl of 10⁶ pfu/ml of Virus 16;    -   50 μl of 10⁵ pfu/ml of Virus 16;    -   50 μl of 10⁷ pfu/ml of Virus 19 (expresses only mouse GM-CSF);    -   50 μl of 10⁶ pfu/ml of Virus 19;    -   50 μl of 10⁵ pfu/ml of Virus 19.

Effects on tumor growth were then observed for a further ≈30 days. Thisexperiment demonstrated superior tumor control and shrinkage with thevirus expressing GALV-R- in both tumor models (see FIG. 14).

EXAMPLE 15 Expression of Two Immune Stimulatory Molecules from a VirusExpressing a Fusogenic Protein

Viruses similar to the GALV-R- and mGM-CSF expressing virus describedabove (Virus 16) were constructed, but additionally expressing mouseversions of CD40L (virus 32), ICOSL (virus 36), OX40L, (virus 35),4-1BBL (virus 33) and GITRL (virus 34). Here, instead of using a plasmidcontainim ICP34.5 flanking regions and an expression cassette comprisingGM-CSF and GALV-R- driven by a CMV and an RSV promoter, a plasmidcontaining ICP34.5 flanking regions and an expression cassettecomprising GM-CSF, GALV and the additional proteins driven by a CMV anRSV and an MMLV promoter respectively were used for recombination with avirus containing GM-CSF, GALV and GFP inserted into ICP34.5. Non-GFPexpressing plaques were again selected. Correct insertion was confirmedby PCR, and expression by western blotting and/or ELISA for theadditional inserted gene. These viruses are shown in FIG. 5. Similarly,viruses expressing anti-mouse and anti-human CTLA-4 in addition to GALVand mGM-CSF were also constructed (Viruses 27 and 31 in FIG. 5 and seealso FIG. 13). Effects of viruses expressing anti-mouse CTLA-4 (virus27), mCD40L, (virus 32), m4-1BBL (virus 33) or mOX40L (virus 35) inaddition to mGM-CSF and GALVR in vivo is shown in FIG. 16 which showedenhanced activity in A20 tumors as compared to virus 16 (expressesmGM-CSF and GALVR-). In these experiments tumors were induced in bothflanks of mice, and virus or vehicle injected only into the right flanktumor. The dose of virus used was 5×10⁴ pfu (50 μl of 1×10⁶ pfu/ml ineach case), given three times over one week. This dose level of virus issubtherapeutic for uninjected tumors for virus 16, which allows thebenefits of the delivery of the additional molecules encoded by viruses27, 32, 33 and 35 to clearly be seen.

Deposit Information

The following HSV1 strains were deposited at the ECACC, CultureCollections, Public Health England, Porton Down, Salisbury, SP4 0JG,United Kingdom on 19 Dec. 2016 by Replimune Limited and were allocatedthe indicated provisional accession numbers:

-   RH004A—Provisional Accession Number 16121902-   RH015A—Provisional Accession Number 16121903-   RH018A—Provisional Accession Number 16121904-   RH021A—Provisional Accession Number 16121905-   RH023A—Provisional Accession Number 16121906-   RH031A—Provisional Accession Number 16121907-   RH040A—Provisional Accession Number 16121908-   RH047A—Provisional Accession Number 16121909.

1. An oncolytic virus which is, or is derived from, a clinical isolatewhich has been selected by comparing the abilities of a panel of threeor more clinical isolates of the same viral species to kill tumor cellsof two or more tumor cell lines in vitro and selecting a clinicalisolate which is capable of killing cells of two or more tumor celllines more rapidly and/or at a lower dose in vitro than one or more ofthe other clinical isolates in the panel.
 2. The vines of claim 1, whichis a modified clinical isolate.
 3. The virus of claim 1, which is aherpes simplex virus (HSV).
 4. The virus of claim 3 which is a HSV1. 5.The virus of claim 1 which is: strain RH018A having the accession numberECCAC 16121904; strain RH004A having the accession number ECCAC16121902; strain RH031A having the accession number ECCAC 16121907;strain RH040B having the accession number ECCAC 16121908; strain RH015Ahaving the accession number ECCAC 16121903; strain RH021A having theaccession number ECCAC 16121905; strain RH023A having the accessionnumber ECCAC 16121906; or strain RH047A having the accession numberECCAC
 16121909. 6. The virus of claim 5 which is strain RH018A havingthe accession number EACC
 16121904. 7. The virus of claim 3, wherein thevirus: (a) does not express functional ICP34.5; (b) does not expressfunctional ICP47; and/or (c) expresses the US11 gene as an immediateearly gene.
 8. The virus of claim 1, wherein the panel comprises 5 ormore different clinical isolates.
 9. The virus of claim 1, wherein thepanel comprises 10 or more different clinical isolates.
 10. The virus ofclaim 1, wherein the panel comprises 20 or more different clinicalisolates.
 11. The virus of claim 1, wherein the tumor cell lines arehuman tumor cell lines selected from the group consisting of U87MG(glioma), HT29 (colorectal), LNCaP (prostate), MDA-MB-231 (breast),SK-MEL-28 (melanoma), Fadu (squamous cell carcinoma), MCF7 (breast),A549 (lung), MIAPACA-2 (pancreas), CAPAN-1(pancreas) and HT1080(fibrosarcoma).
 12. The virus of claim 1, which comprises: (a) one ormore immune stimulatory molecules or one or more immune stimulatorymolecule encoding genes; and/or (b) one or more fusogenicprotein-encoding genes.
 13. The virus of claim 12, wherein: (a) thefusogenic protein is selected from the group consisting of vesicularstomatitis virus (VSV) G-protein, syncitin-1, syncitin-2, simian virus 5(SV5) F-protein, measles virus (MV) H-protein, MV F-protein, respiratorysyncytial virus (RSV) F-protein and a glycoprotein from gibbon apeleukemia virus (GALV), murine leukemia virus (MLN), Mason-Pfizer monkeyvirus (MPMV) or equine infectious anaemia virus (EIAV) from which the Rpeptide has been deleted; and/or (b) the immune stimulatory molecule isGM-CSF, IL-2, IL-12,IL-15, IL-18, IL-21, IL-24, a type I interferon,interferon gamma, a type III interferon, TNF alpha, an antagonist of TGFbeta, an immune checkpoint antagonist or an agonist of an immunepotentiating pathway including CD40 ligand (CD40L), ICOS ligand, GITRligand, 4-1-BB ligand, OX40 ligand or flt3 ligand or a modified versionof any of these.
 14. The virus of claim 1, wherein: (a) the fusogenicprotein is the glycoprotein from gibbon ape leukemia virus (GALV) andhas the R transmembrane peptide mutated or removed (GALV-R-); and/or (b)the immune stimulatory molecule is GM-CSF, GITRL, ICOSL, 4-1-BBL, OX40Lor CD40L, or a modified version thereof, or the immune stimulatorymolecules are GM-CSF and GITRL, OX40L, 4-1-BBL, ICOSL or CD40L, or amodified version thereof.
 15. The virus of claim 11, wherein: (a) thefusogenic protein-encoding gene and/or the immune stimulatorymolecule-encoding gene are inserted into the ICP34.5 encoding locus,either by insertion, or partial or complete deletion, each underseparate regulatory control, optionally in a back to back orientation inrelation to each other; and/or (b) the sequence of the gene encoding thefusogenic protein and/or the sequence of the gene encoding the immunestimulatory molecule is codon optimized so as to increase expressionlevels in target cells.
 16. A virus which expresses three heterologousgenes, wherein each of the three heterologous genes is driven by adifferent promoter selected from the CMV promoter, the RSV promoter, theSV40 promoter and a retroviral LTR promoter.
 17. The virus of claim 1,which expresses three heterologous genes, wherein each of the threeheterologous genes is driven by a different promoter selected from theCMV promoter, the RSV promoter, the SV40 promoter and a retroviral LTRpromoter.
 18. The virus of claim 16, which expresses four heterologousgenes driven by each of the CMV promoter, the RSV promoter, the SV40promoter and a retroviral LTR promoter, respectively.
 19. The virus ofclaim 16, where the retroviral LTR is from MMLV.
 20. A virus whichexpresses three heterologous genes, wherein each of the threeheterologous genes is terminated by a different poly adenylationsequence selected from the BGH, SV40, HGH and RBG poly adenylationsequences.
 21. The virus of claim 1, which expresses three heterologousgenes, wherein each of the three heterologous genes is terminated by adifferent poly adenylation sequence selected from the BGH, SV40, HGH andRBG poly adenylation sequences.
 22. The virus of claim 20, whichexpresses four heterologous genes terminated by each of the BGH, SV40,HGH and RBG poly adenylation sequences, respectively.
 23. The virus ofclaim 16 which is (a) a HSV; (b) a HSV1; or (c) a pox virus.
 24. Apharmaceutical composition comprising the virus of claim 1 and apharmaceutically acceptable carrier or diluent. 25.-34. (canceled)
 35. Aproduct of manufacture comprising a virus according to claim 1 in asterile vial, ampoule or syringe.
 36. A method of treating cancer, whichcomprises administering a therapeutically effective amount of the virusof claim 1 to a patient in need thereof.
 37. The method of claim 36,which further comprises administering a therapeutically effective amountof a further anti-cancer agent to a patient in need thereof.
 38. Themethod of claim 37, wherein the further anti-cancer agent is selectedfrom an antagonist of an immune co-inhibitory pathway, an agonist of animmune co-stimulatory pathway, radiation and/or chemotherapy, an agentthat targets a specific genetic mutation which occurs in tumors, anagent intended to induce an immune response to one or more tumorantigen(s) or neoantigen(s), a cellular product derived from T cells orNK cells, an agent intended to stimulate the STING, cGAS, TLR or otherinnate immune response and/or inflammatory pathway, a second virusoptionally an oncolytic virus, an inhibitor of the indoleamine2,3-dioxygenase (IDC)) pathway, and combinations thereof.
 39. The methodof claim 38, wherein the antagonist of an immune co-inhibitory pathwayis a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3inhibitor, a TIM-3 inhibitor, a VISTA inhibitor, aCSE1R inhibitor, anIDO inhibitor, a CEACAM1 inhibitor, a KIR inhibitor, a SLAMF7 inhibitoror a CD47 inhibitor, and/or the agonist of an immune co-stimulatorypathway is a GITR agonist, a 4-1-BB agonist, an OX40 agonist, a CD40agonist or an ICOS agonist.
 40. The method of to claim 38, wherein thefurther anti-cancer agent comprises an antibody.
 41. The method of claim37, wherein the virus and the further anti-cancer agent(s) areadministered separately.
 42. The method of claim 37, wherein the virusand the further anti-cancer agent(s) are administered concurrently. 43.The method of claim 37, wherein the cancer is a solid tumor. 44.-45.(canceled)
 46. A method of selecting an oncolytic virus, the methodcomprising: (i) comparing the abilities of a panel of three or moreclinical isolates of the same viral strain to kill tumor cells of two ormore tumor cell lines in vitro; (ii) scoring the abilities of each ofthe panel of viruses to kill tumor cells; (iii) selecting a virus whichhas one of the best scores; (iv) optionally modifying the virus toinactivate one or more viral genes; and/or (v) optionally modifying thevirus to express one or more immune stimulatory molecule encoding genesand/or one or more fusogenic protein-encoding genes.